Method for diagnosing a disease state using ocular characteristics

ABSTRACT

A method for diagnosing a disease state in a subject, including examining the subject&#39;s eyes in order to determine whether the subject exhibits one or more symptoms of the disease state. Symptoms of the disease state include altered ocular characteristics, such as pupil size, pupil motility, ocular blood vessel coloration, and strobe light cutoff frequency. By evaluating changes to ocular characteristics, a disease state can be diagnosed in a subject. In addition, the severity of the disease state also can be determined.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/740,979, filed Dec. 19, 2003, now U.S. Pat. No. 7,703,918B2, which claims priority to U.S. Provisional Application No. 60/434976filed Dec. 19, 2002.

This invention was made in part with government support under contractnumber DAAH01-02-C-R171 awarded by the Defense Advanced ResearchProjects Agency (DARPA). The government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to ocular characteristics and their role inassessing physical well-being and diagnosing the existence and severityof a disease state.

2. Related Art

The use of chemical and biological agents is an ever-increasing threatto people of all nations. In order to properly treat those who have beenexposed to biological or chemical weapons, a rapid diagnosis isimperative. Additionally, in instances where there have been masscasualties as a result of exposure to chemical agents or biologicaltoxins, there is a need for a system of rapidly assessing the severityof each individual's exposure in order to determine the requirements forappropriate treatment.

Due to the interconnection between the eyes and the variousphysiological systems of the body (e.g., cardiovascular,musculoskeletal, lymphatic, etc.), the eyes may be a key to rapidlydiagnosing disease states, including biological toxin or chemical agentexposure. U.S. Pat. No. 6,305,804 issued to Rice et al. discloses amethod for detecting the concentration of blood components, such ashemoglobin, glucose, and bilirubin. While this information may be usefulto a person already diagnosed with a disease state, i.e., diabetes, formonitoring his or her glucose levels, or to diagnose jaundice in aninfant, it does not provide a method for diagnosing any other diseasestates, such as exposure to chemical agents or biological toxins.Likewise, Rice et al. does not disclose a method for assessing theseverity of exposure to such an agent or toxin.

U.S. Pat. No. 6,626,537 issued to Odom et al. discloses a method formonitoring a medical condition by analyzing light reflected back from asubject's eyes. Using the method taught by Odom et al., it may bepossible to determine whether an abnormality exists in the patient'seyes. However, Odom does not teach a method for identifying, i.e.,diagnosing, an unknown disease state. Nor does Odom teach a method forassessing the severity of a disease state previously identified. Odom etal. therefore does not provide a method for diagnosing and evaluatingthe severity of exposure to a biological toxin or chemical agent. Therethus remains a need for a real-time, non-invasive method for diagnosingnot only the existence of a disease state but also the severity, therebyallowing treatment strategies to be prioritized with respect to need,especially in situations involving mass casualties.

SUMMARY OF THE INVENTION

The present invention solves the foregoing problems by providing anon-invasive real-time method for diagnosing a disease state in asubject based upon ocular characteristics.

Through temporal assessment of ocular characteristics, the existence andseverity of a disease state can be diagnosed.

One aspect of the invention is a method of diagnosing a disease state ina subject, including the steps of examining the subject's eye in orderto determine whether the subject exhibits an ocular characteristic ofthe disease state, and if so, evaluating the ocular characteristic inorder to diagnose the disease state, including its severity.

Another aspect of the invention is a method of diagnosing cyanideexposure of a subject, including the step of determining whether thesubject's ocular blood vessel coloration varies significantly from anormal ocular blood vessel coloration.

Another aspect of the invention is a method of diagnosing carbonmonoxide exposure of a subject, including the step of determiningwhether the subject's ocular blood vessel coloration variessignificantly from normal ocular blood vessel coloration.

Another aspect of the invention is a method of diagnosing botulinumtoxin exposure of a subject, including the step of determining whetherthe subject's strobe cutoff frequency varies significantly from a normalstrobe cutoff frequency.

Another aspect of the invention is a method of diagnosing exposure to achemical agent or a biological toxin, including the steps of evaluatinga primary ocular characteristic to determine whether there has beenexposure to the chemical agent or biological toxin, and evaluating asecondary ocular characteristic to determine to what extent the subjectwas exposed to the chemical agent or biological toxin.

A feature of the invention is a method for diagnosing a disease state ina subject based upon altered ocular characteristics in the subject.

An advantage of the invention is that the eye examination can beperformed non-invasively.

Another advantage of the invention is that ocular characteristics can beused to rapidly and accurately diagnose individuals who have beenexposed to chemical agents or biological toxins, including but notlimited to: organophosphate compounds (anti-cholinesterases), cyanidecompounds, carbon monoxide, and botulinum toxin.

Another advantage of the invention is that it provides a real-timeassessment which may serve as an early-warning system for possiblecomplications based upon generalized information obtained from ocularcharacteristics.

BRIEF DESCRIPTION OF THE DRAWINGS

The application contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee.

The present invention is described with reference to the accompanyingdrawings. In the drawings, like reference numbers indicate identical orfunctionally similar elements. Additionally, the left-most digit(s) of areference number identifies the drawing in which the reference numberfirst appears.

FIG. 1 is a flow chart showing an exemplary embodiment of a method fordiagnosing exposure to a biological agent or chemical toxin.

FIG. 2 is a concentration-response curve for Sprague-Dawley rats 5minutes after direct ocular exposure to parathion.

FIG. 3 is a concentration-response curve for Sprague-Dawley rats 5minutes after direct ocular exposure to parathion.

FIG. 4 is a fundoscopic image of a Sprague-Dawley rat eye (A) prior to,and (B) 2 minutes after subcutaneous injection 10 mg/kg KCN (fadedcircular object in the center of each image is a reflection of the lightsource from the fundoscope).

FIG. 5 shows the percent change in arterial and venous vessel colorationin Sprague-Dawley rats in response to subcutaneous injection of 1 mg/kgKCN over time, n=6. Percent changes are compared to pre-dosingindividual control levels. Change in venous coloration becamestatistically significant (p<0.05) at the 90 second time interval. Meanshave been normalized to initial control values and error bars represent±S.E.M.

FIG. 6 shows the percent change in venous vessel coloration inrepresentative Sprague-Dawley rats in response to subcutaneous injectionof various concentrations of KCN over time. Percent changes are comparedto pre-dosing individual control levels (normalized to control). Errorbars have been omitted for clarity.

FIG. 7 shows a concentration-response curve for venous colorationchanges in Sprague-Dawley rats in response to subcutaneous injection ofKCN. Data was obtained 5 minutes after KCN injection. Extrapolated EC₅₀=24.6±3.1 μg/kg. Percent changes are compared to pre-dosing individualcontrol levels. Means have been normalized to the maximal observedchange in all animals (53%) and error bars represent ±S.E.M.

FIG. 8 shows the percent change in arterial vessel coloration(normalized to control) in representative Sprague-Dawley rats inresponse to inhalational application various levels of carbon monoxideover time. Error bars have been omitted for clarity.

FIG. 9 shows the percent change in arterial and venous vessel coloration(normalized to control) in Sprague-Dawley rats in response toinhalational application of 3000 ppm CO over time, n=6. Changes inarterial and venous coloration became statistically significant (p<0.05)at the 15 minute and 30 minute time intervals, respectively.

FIG. 10 is a concentration-response curve for fundal artery colorationchanges in Sprague-Dawley rats in response to inhalational exposure tocarbon monoxide gas for 60 minutes. Data was obtained 60 minutes afterinitiation of carbon monoxide administration except for one animal fromthe 2500 ppm group which expired prior to this time point. Data for thisanimal was obtained from images taken approximately 1 minute prior torespiration cessation (after 51 minutes of carbon monoxide inhalation).Extrapolated EC₅₀=289±37 ppm. Changes became statistically significant(p<0.05) at the 250 ppm for 60 minutes dose. Means have been normalizedto the maximal observed change in all animals (38.5%) and error barsrepresent ±S.E.M.

FIG. 11 is a time-course plot for the occurrence of symptomology anddeath for Sprague-Dawley rats injected (I.P.) with varying doses ofbotulinum toxin. Data are plotted as means, error bars are omitted tosimplify graph. Data points which had means of greater than 48 hours arenot plotted. Y-axis ([Botulinum Toxin], pg/kg) is logarithmic andbase-10 notation is used to indicate dose in picograms/kilogram. Loss ofexploratory behavior was used for quantitation of general motorsymptomology (see text). Significant (p<0.05) decrease in pupillaryreflex to increased light intensity (compared to baseline) used toquantitate impaired pupillary reflex. Note: larger Y-axis values are atthe bottom of the graph.

FIG. 12 is a concentration-response curves for Sprague-Dawley ratsexposed to various concentrations of botulinum toxin, I.P. Data areplotted as means ±S.E.M. Data points which had means of greater than 48hours are not plotted. X-axis ([Botulinum Toxin], pg/kg) is logarithmicand base-10 notation is used to indicate dose in picograms/kilogram.Loss of exploratory behavior was used for quantitation of general motorsymptomology (see text). Significant (p<0.05) decrease in pupillaryreflex to increased light intensity (compared to baseline) used toquantitate impaired pupillary reflex. See text for description ofdecreased strobe accommodation frequency. Decrease was indicated whenvalue was significantly (p<0.05) less than control (i.e., 3.1 Hz orless). Note: larger X-axis values are at the left of the graph.

EMBODIMENTS OF THE INVENTION

Ocular Characteristics

An ocular characteristic is a measurable physical attribute determinedvia the observation of external and/or internal features of the eye(s).Non-limiting examples of ocular characteristics include pupil size,pupillary light reflex, pupil motility, strobe light frequency cutoff,blood vessel coloration, blood vessel architecture, ischemic spots,exudate appearance, and cellular degeneration. An altered ocularcharacteristic can be the result of a disease state. In many instances,an altered ocular characteristic is specific for a particular diseasestate. This need not be the case, however, for the method of the presentinvention to be useful, as it is the cumulative evaluation of multipleocular characteristics which often results in a specific diagnosis. Inaddition, particular diseases often can be eliminated from adifferential diagnosis based upon the subject's medical history. Forexample, while a soldier deployed in combat and a worker in a paintfactory may both exhibit the same altered ocular characteristics, adifferent diagnosis can be reached based upon the risk factorsassociated with the respective environments to which each is exposed.

Disease State

The term “disease state,” optionally referred to simply as “disease,” isintended to encompass not only the meaning as commonly understood bythose skilled in the art to which this invention belongs but alsoconditions not necessarily pathological in nature. For purposes of thisinvention, a disease state is any condition the existence of whichresults in an alteration to one or more ocular characteristics in thesubject. Non-limiting examples of a disease state include biologicaltoxin or chemical agent exposure, systemic hypertension, internaltrauma, inadequate nutritional status, and altered cognitive state.Biological toxins and chemical agents include, but are not limited to:organophosphates, cyanide, carbon monoxide, and botulinum toxin.

Organophosphates

Many organophosphates (OPs), including tabun, sarin, soman, syclosarin,and VX, are nerve agents that can be used for chemical warfare. Othermembers of the OP group are commonly used as agricultural pesticides(e.g., Malathion, Parathion, Trichlorfon, etc.). The OPs share a similarchemical structure and are derivatives of phosphoric or phosphonic acidscontaining two alkyl groups and a leaving group. The primarypharmacological effect of the OPs is to inhibit the enzymeacetylcholinesterase (AChE) via phosphorylation of a serine hydroxylgroup in the active site of the enzyme. AChE normally functions toquickly destroy acetylcholine (ACh) in the synaptic cleft and thusterminate transmission of a nerve impulse. Inactivation of AChE resultsin the accumulation of ACh, which causes an initial enhancement andprolongation of cholinergic activity followed by depolarizationblockade.

Cyanide

The cyanides, when compared to other lethal chemical warfare agents, arethe least toxic. However, though these agents may be less potent intheir lethality, they act very quickly and thus diagnosis and subsequentactions to combat their effects must commence promptly after exposure.The cyanides of military interest are the volatile liquids: hydrogencyanide (HCN), cyanogen chloride (ClCN), and cyanogen bromide (BrCN).The pharmacological and toxicological effects of the cyanides (due tocyanide [CN⁻]) are identical, but the cyanogens are additionallyirritant to the eyes and respiratory tract. Cyanide acts by combiningwith the ferric ion in mitochondrial cytochrome oxidase, preventingelectron transport in the cytochrome system and, thus, bringingoxidative phosphorylation and ATP production to a halt. Though fullyoxygenated, the cells cannot utilize the oxygen and thus increaseddemands are placed on anaerobic glycolysis. This results in lactic acidproduction/accumulation and eventual cell death due to histotoxicanoxia.

Carbon Monoxide

Carbon monoxide (CO) is a colorless, odorless gas which comes fromnumerous sources. Part of CO's acute toxicity results from the fact thatit essentially replaces the oxygen in the blood. Hemoglobin (Hgb), theoxygen carrying molecule of the blood, has an affinity for CO which isapproximately 200-250 times greater than that for oxygen. The absorptionof CO by the blood (creating carboxyhemoglobin) causes a leftward shiftin the oxygen-hemoglobin dissociation curve, resulting in a decreasedoxygen carrying capacity and impaired release of oxygen to the tissues.This, in turn, leads to cellular/tissue hypoxia. The relatively highaffinity of Hgb for CO also results in a relatively persistent effect inindividuals well after they've been removed from exposure to the gas.

Though. carbon monoxide poisoning generally results from chronicexposure to lower levels of CO, there are many documented cases of acutepoisoning. In addition, the relatively low level of toxicity andreversibility of CO poisoning has allowed more extensive human studiesto be performed than with many other toxins. The brain and heart, byvirtue of their high metabolic activity, appear to be the most rapidlyaffected sites of CO poisoning. Unfortunately, many of the early warningsigns for CO poisoning are similar to common flu-like symptoms:headache, nausea, dizziness, etc. In cases of more severe poisoning, theclinical findings may be very similar to that of cyanosis (cyanidepoisoning; see above). This is somewhat to be expected in that bothcarbon monoxide and cyanide deprive tissues/cells of the ability toutilize oxygen. However, the different pathways by which these toxinsreach this physiological endpoint may be of use in the quickdetermination of the type of poison in the system. As described above,for cyanide poisoning the hemoglobin/blood is fully oxygenated but thecells are deprived the ability to utilize this oxygen. On the otherhand, with CO poisoning the hemoglobin/blood is severelyunder-oxygenated due to the formation of carboxyhemoglobin. This alteredlevel of blood oxygenation is discernable based upon the coloration ofthe blood which is primarily determined by hemoglobin and its level ofoxygenation.

Hemoglobin (Hgb), the red blood pigment, provides the oxygen-carryingcapacity of the blood. The high concentration (34 gm/dL) of Hgb withinerythrocytes (red blood cells) allows oxygen (O₂) to be circulated atconcentrations as high as 0.01 M, which is approximately the same asatmospheric air. This oxygen concentrating ability is accomplished via anumber of factors. Hgb is composed of four subunits, each of whichnoncovalently binds a single heme group. The heme molecule, which isresponsible for the characteristic red color of blood, is the site atwhich each Hgb subunit binds one molecule of O₂. Thus, each Hgb moleculemay bind four molecules of O₂ (one per subunit). Furthermore, thisoxygen is bound cooperatively. That is, when the Hgb binds a molecule ofO₂, its affinity for binding another molecule of O₂ is increased. Thus,the fourth O₂ molecule to bind Hgb does so with 100-fold greateraffinity than did the first. The reverse is also true. As oxygen beginsto unbind upon reaching the tissues, the affinity for the still-bound O₂decreases such that each successive molecule more easily dissociates.This simple property of Hgb provides the foundation for highly efficientloading of oxygen in the lungs and unloading in the tissues. In additionto its role as an O₂ carrier, Hgb also plays an important role in thetransport of CO₂, acquiring it in the tissues and subsequently releasingit in the lungs for expiration.

Botulinum Toxin

Botulinum toxin is the most poisonous substance known to man with asingle gram of the crystalline toxin possessing the ability to kill morethan 1 million people. Because of this extreme potency and lethality, aswell as its ease of production and transport, botulinum toxin poses amajor bioweapons threat. Botulinum toxin is a protein neurotoxinproduced by various subtypes of the bacterium Clostridium botulinum, ananaerobic, gram-positive organism. Unable to penetrate intact skin,botulinum toxin requires a wound, ingestion, or inhalation to exert itseffects. Upon gaining access to the general circulation, botulinum toxinbinds with extremely high affinity to peripheral cholinergic nerveendings at the neuromuscular junction and in the autonomic nervoussystem (preganglionic sympathetic and parasympathetic, postganglionicparasympathetic nerve terminals). Once in the target cell, the toxinacts as a zinc-dependent endoprotease, cleaving polypeptides that areessential for the exocytosis (release) of acetylcholine (ACh). Botulinumtoxin is both highly selective for cholinergic neurons and longlasting—clinically significant responses may last from several months toa year.

The human data concerning botulinum toxin effects comes primarily fromcases of food-borne (ingested) and wound exposure. The classic clinicalfeatures of botulism (the disease caused by botulinum toxin exposure)are symmetric cranial neuropathies (i.e., ptosis [drooping eyelids],weakened jaw clench, dysarthria [speech disturbances], and dysphagia[difficulty swallowing]), diplopia (double-vision) or blurred vision,peripheral muscle weakness, respiratory dysfunction, andgastrointestinal distress (nausea and vomiting). In addition, pupillarylight reflexes are typically depressed or absent and the pupils aredilated (mydriasis). With time, peripheral muscle weakness will progressto flaccid muscular paralysis and death results from respiratory muscleparalysis. Though little is published regarding the bioterrorist ormilitary use of botulinum toxin, it is believed that such an attack(food-borne or aerosolized) would yield similar symptoms presenting12-72 hours after exposure. In the case of inhalational exposure, theclinical presentation would be identical but the gastrointestinalsymptoms would likely be absent.

Method of Diagnosing a Disease State

The present invention includes a method of diagnosing a disease state ina subject. The subject can be any member of the animal kingdom, such asdogs, monkeys, rats, etc., but preferably is a human being (person). Themethod of diagnosing the disease state preferably includes the stepsof 1) examining the subject's eyes in order to determine whether thesubject exhibits an ocular characteristic indicative of the diseasestate, and if so; 2) evaluating the ocular characteristic in order todiagnose the disease state, including its severity. One or more alteredocular characteristics can indicate the existence of a disease state ina subject.

In a presently preferred embodiment, a subject's eyes are examinedpassively rather than actively. A passive examination is distinguishedfrom an active examination based upon the degree of participation by thesubject. During a passive examination, the examiner may manipulate theeyes with lighting, etc., but the subject is not required to voluntarilyact or react to external stimuli. In contrast, the subject doesparticipate during a typical active examination, for example by trackingwith his or her eyes a light or the examiner's finger. The subject'seyes preferably are examined by evaluating one or more ocularcharacteristics. For example, it may be desirable to examine a subject'seyes by sequentially evaluating pupil size, pupillary light reflex,ocular blood vessel coloration, and strobe light cutoff frequency. Bysequentially analyzing a variety of ocular characteristics, a diagnosiscan be made either manually by a caregiver, or automatically, i.e.,without the need for manual measurement, analysis, or diagnosis, by aprocessing unit or other similar means employing a decision tree or analgorithm.

In a presently preferred embodiment, ocular characteristics of asubject's eye are evaluated by first quantifiying the ocularcharacteristic under consideration. For example, the diameter of thesubject's pupil can be measured and assigned a numerical charactercorresponding to the diameter. This number, or quantity, can then becompared easily to a normal pupil under similar conditions, which alsohas been quantified by assigning an appropriate numerical character. Thenorm for the ocular characteristic, in the case of this example thenormal pupil size, preferably is established for each subject beingexamined. However, average normal values also can be established fordifferent populations and sub-populations. A subject's quantified ocularcharacteristic data can then either be compared to his or her personalnormal value, or it can be compared to an average normal valueestablished for a population to which the subject belongs. In so doing,a person examining a subject's eye can determine whether one or moreocular characteristics have been altered.

Pupil Size

Referring generally to FIG. 1, pupil size preferably is one of theocular characteristics evaluated when examining the subject's eyes. Thepupil(s) preferably are evaluated to assess whether there has been asignificant increase (dilation) or decrease (miosis) in the diameter ofthe pupil(s). Non-limiting examples of times at which it may bedesirable to analyze a subject's pupil size are after suspected orpossible exposure to a biological toxin or chemical agent, when thesubject displays symptoms of or is at risk for developing systemichypertension, when there is a possibility that the subject has suffereda blunt head injury, as part of a routine check-up or health screening,and after the occurrence of any other event likely to induce a diseasestate in the subject.

In order to determine whether there has been a significant change inpupil size, a baseline, or “normal,” pupil size preferably isestablished. A normal pupil size preferably is established for eachsubject by measuring the diameter of each subject's pupil(s) under acontrolled setting, i.e., controlled lighting, etc. Alternatively, anaverage (or baseline) pupil size for a given population to which thesubject belongs can be used as the normal pupil size for all members ofthe population. While examining a subject's eye, the diameter of thesubject's pupil preferably is measured. The subject's pupil(s)preferably are evaluated in two settings—one with low intensity light(for example about 1.7 cd/m²), and the second with high intensity light(for example about 80 cd/m²). As shown in FIG. 1, a significant decreasein pupil size (pupil contraction or “miosis”) in low intensity light canindicate organophosphate exposure.

The extent of organophosphate exposure can be evaluated by examiningwhether the subject's pupil(s) retain their ability to contract anddilate in response to altering light conditions (pupillary lightreflex). Pupil(s) retain their pupillary light reflex when subjects areexposed to relatively low levels of an organophosphate. The pupillarylight reflex is eliminated, however, when subjects are exposed torelatively high levels of an organophosphate. After organophosphateexposure is diagnosed, the extent of the exposure can be determined byrepeatedly exposing the affected subject's pupil(s) to a high intensitylight followed by a low intensity light. Those subjects in which thepupillary light reflex has been eliminated (or greatly reduced) havebeen exposed to relatively high levels of an organophosphate. Incontrast, those subjects in which the pupillary light reflex remainsintact have been exposed to relatively low levels of an organophosphate.This aspect of the invention can be especially useful as a means fortriaging casualties in military field operations where masses ofmilitary personnel can possibly be exposed to chemical warfare agents atthe same time. As shown in FIG. 1, a significant increase in pupil size(dilation) in low intensity light can indicate possible cyanide orbotulinum toxin exposure. Pupil dilation in high intensity light is afurther indicator of possible botulinum toxin exposure.

A subject's pupil(s) can be examined using various means commerciallyavailable and known to those skilled in the art. In a presentlypreferred embodiment, the subjects' pupil(s) are examined using infraredand visible frequency adjustable lighting and adequate resolution blackand white infrared and color near-infrared CCD (charge-coupled device)and color visible wavelength cameras.

Ocular Blood Vessel Coloration

Ocular blood vessel coloration preferably is another ocularcharacteristic evaluated when examining a subject's eye. Ocular bloodvessel coloration preferably is examined to assess whether there hasbeen a significant change, e.g., increased brightness or darkness, ofthe blood in the ocular blood vessels. Non-limiting examples of times atwhich it may be desirable to analyze a subject's pupil size are aftersuspected or possible exposure to a biological toxin or chemical agent,when the subject displays symptoms of or is at risk for developingsystemic hypertension, when there is a possibility that the subject hassuffered a blunt head injury, as part of a routine check-up or healthscreening, and after the occurrence of any other event likely to inducea disease state in the subject. Ocular blood vessels include, but arenot limited to: arteries, veins, venules, capillaries, and arterioles.

In order to determine whether there has been a significant change inocular blood vessel coloration, a baseline, or “normal,” colorpreferably is first established for certain ocular blood vessels, e.g.,retinal veins and arteries. Normal coloration preferably is establishedfor each subject using a modified fundoscope for imaging the internalregions of the eye under non-mydriatic conditions. Alternatively, anaverage (or baseline) ocular blood vessel coloration for a givenpopulation to which the subject belongs can be used as the normal vesselcoloration for all members of the population. While examining thesubject's eye, fundoscopic images of the internal regions of thesubject's eyes can be obtained for comparison purposes. As shown in FIG.1, a significant increase in the brightness of the retinal veins canindicate possible cyanide exposure. Significantly increased brightnessof the retinal veins, combined with an increase in pupil size in lowintensity light, can lead to a definitive diagnosis of cyanide exposure.In contrast, a significant decrease in brightness of the retinal veinscan indicate possible carbon monoxide exposure. A diagnosis of carbonmonoxide exposure can be confirmed by analyzing retinal arterycoloration. A significant decrease in brightness of the retinalarteries, combined with a significant decrease in retinal veinbrightness, can lead to a definitive diagnosis of carbon monoxideexposure.

Arterial coloration also can be useful as a secondary characteristic forassessing the severity of cyanide exposure. Whereas retinal veincoloration is affected at relatively low doses of cyanide exposure,retinal artery coloration is not affected until the subject has beenexposed to relatively high doses of cyanide. This differentialsensitivity of the retinal veins and arteries is a useful method fordetermining the severity of cyanide exposure. A similar phenomenonexists with carbon monoxide exposure. Whereas retinal artery colorationis affected at relatively low doses of carbon monoxide exposure, retinalvein coloration is not affected until the subject has been exposed torelatively high doses of carbon monoxide: An analysis of thedifferential response of retinal arteries and veins thus also can beuseful for determining the severity of carbon monoxide exposure.

Ocular blood vessels, including retinal arteries and veins, can beexamined using various means known to those skilled in the art, such asa fundoscope. The fundoscope preferably can be converted to a digitalvideo fundoscope by making modifications such as: altering shuttermechanics, adapting the mounting means to allow a CCD camera to beattached, and refocusing/magnifying the light source and image capturemeans to provide fundoscopic images with increased clarity.Alternatively, a commercially available digital fundoscope could beused.

Strobe Cutoff Frequency

Strobe light cutoff frequency preferably is another ocularcharacteristic evaluated when examining a subject's eye. In normal,i.e., healthy or disease-state-free, subjects, there is a maximalfrequency at which the pupillary light reflex can function. Thisfrequency is limited by the frequency at which impulses denoting a lightflash can travel from the retina, through the pupillary light reflexloop, and back to the muscles controlling pupil size for effect. Forcomparison purposes, this value is considered the normal (or baseline)strobe light cutoff frequency. The normal strobe cutoff frequencypreferably is determined empirically for each subject. Alternatively, anaverage (or baseline) cutoff frequency for a population to which thesubject belongs can be determined and used.

At a pre-determined time, strobe cutoff frequency is obtained forcomparison purposes. A significant decrease in the maximal responsefrequency after over-stimulation with high intensity light flashes canindicate botulinum toxin exposure. Strobe cutoff frequency preferably isexamined using a tunable strobe device for applying various intensitiesof light at known frequencies.

EXAMPLES

The following examples are from studies employing a rodent model(Sprague-Dawley rat) of exposure.

Example 1 Intraperitoneal Exposure to Organophosphates

Parathion is an organophosphate insecticide which has an identicalmechanism of action to that of the organophosphate nerve agents (e.g.,Soman, Sarin, VX, etc.) but has decreased potency compared to thewarfare agents. Parathion was diluted in corn oil as necessary fordosing. The literature LD₅₀ values (dose required to kill 50% of thoseanimals exposed) were first validated for parathion. The literaturevalue for rats exposed to intraperitoneal (I.P.) parathion is 2 mg/kg(i.e., 2 mg parathion per kg of rat body weight). The findings in thepresent study were consistent with this value—extrapolated LD₅₀ valuefrom concentration-response (death) curves was 2.47±0.67 mg/kg(±standard deviation). The lowest dose found to be lethal in any animaltested within 48 hours of administration (standard time length for LD₅₀testing) was 1.00 mg/kg. This occurred for only 1 of 5 animals tested atthis dose.

Upon validation of the literature LD₅₀, a concentration-response curvefor miosis (pupillary contraction) at various times post-exposure wasconstructed. Animals were weighed and then briefly anesthetized with theinhalational anesthetic halothane (Sigma-Aldrich, St. Louis, Mo.) andgiven an I.P. injection of parathion (Supelco, Bellefonte, Pa.). Theparathion stock concentration was chosen for each group of animals suchthat the total volume administered to each animal was from 0.2 to 1.0ml. The data discussed below is in relation to individual controls(i.e., control pupil diameter values were obtained for each animal priorto injection of parathion). For proper control purposes, however, agroup of animals (n=7) was given 1 ml corn oil I.P. to ensure lack ofmiotic effect of the vehicle (percent change in pupil size 60 minutesafter injection=0.28±0.97%).

Test animal pupils were monitored and recorded continuallypost-injection of parathion for a period of 60 minutes under very lowlight intensity conditions (1.7 cd/m²) using CCD cameras (color andinfrared). At specific time points during these recordings (5, 10, 15,30, and 60 minutes), the animals were subjected to “steps” in lightintensity for pupillary light response analysis. For each time pointindicated, this involved 1 minute of high intensity lighting (80 cd/m²)followed by return to baseline low level lighting intensity. Though therestricted time analysis may not allow for full dark/light adaptation,it did supply sufficient responses for temporal analysis of earlypupillary light reflexes around each time point investigated andprovided more opportunities to examine light reactivity than would theanalysis of full adaptive responses. The concentration-response curve(for miosis/percent pupillary contraction) 15 minutes post-parathioninjection is illustrated in FIG. 2, in which all values have beennormalized to the maximal pupillary contraction observed among allparathion exposed animals (93.1%; 3 μg/kg), light intensity=1.7 cd/m²,error bars represent ±S.E.M, and extrapolated EC₅₀ is 147±36 ng/kg.

The pupillary light reflex was also analyzed in these animals. Thoughthis reflex (contraction of pupil in response to increased lightintensity and dilation of the pupil in response to decreased lightintensity) was altered and sometimes absent in animals at lower doses ofparathion, it was not completely absent for all animals until the dosingreached a level of 30 mg/kg (EC₅₀ for loss of pupillary lightreflex=953±69 μg/kg). This differential potency of organophosphates formiosis and abolition of the light reflex thus can serve as a beneficialdiagnostic tool.

Miosis is a relatively sensitive ocular characteristic fororganophosphate exposure with a diagnostic index (ratio of miosis EC₅₀to compound LD₅₀) of approximately 200 (e.g., occurs at doses 200-foldless than those found to be lethal) in the case of I.P. injection andapproximately 14,000-fold for direct ocular exposure. In addition, thefact that loss of the pupillary light reflex occurred at slightlygreater levels of organophosphate intoxication than did miosis, providesan additional diagnostic tool for use during ocular examination. Thatis, not only may ocular analysis serve to diagnose whether ananimal/individual has been exposed, but it may also provide informationas to the level of exposure. Such differential diagnosis with regard tothe severity of exposure may prove extremely useful in possible triagecases where large numbers of individuals have been exposed to variouslevels of agent and categorizing individuals with respect to medicalneed is vital.

Example 2 Direct Ocular Exposure to an Organophosphate

In addition to the intraperitoneal studies described above, groups ofanimals were also exposed to direct ocular parathion to better simulatelikely warfare exposure (vapor exposure to the eyes and respiratorytract). These studies were conducted identically to those describedabove except that rather than I.P. injection, the left eye was exposedto 0.05 ml of the parathion concentration for a period of 3 minutes andthen washed with saline. During the application period, animals wereheld under light (1%) halothane anesthesia to prevent unwanted movementsor blinking which might alter the application time/integrity. Timespost-exposure represent the amount of time elapsed from initialparathion application (i.e., parathion was washed off at 3 minutespost-exposure).

FIG. 3 shows the concentration-response curve for 5 minutes post-directocular exposure. The earlier time point was chosen for these studies asmiosis developed much more rapidly via exposure by this route. Similarto the findings for I.P. parathion, the loss of the pupillary lightreflex occurred at slightly higher concentrations than did miosis. Thefirst group of animals in which all lacked such a response was the 10μg/kg group with an EC₅₀ for loss of the light reflex equal to 2.8±0.9μg/kg. In FIG. 3, all values have been normalized to the maximalpupillary contraction observed among all parathion exposed animals(93.1%; 3 μg/kg). Light intensity=1.7 cd/m². Error bars represent±S.E.M. Extrapolated EC₅₀ is 147±36 ng/kg.

Example 3 Exposure to Cyanide

The LD₅₀ for potassium cyanide (KCN; Sigma-Aldrich, St. Louis, Mo.) wasvalidated. using subcutaneous KCN. The experimental LD₅₀ value forsubcutaneous KCN was 6.7±0.6 mg/kg compared to literature values rangingfrom 6 to 9 mg/kg. Studies were done with both anesthetized (withKetamine/Xylazine, Sigma-Aldrich, St. Louis, Mo.) and unanesthetizedanimals. No differential effects were observed. Animals that survivedthe first two hours after KCN exposure, survived for the duration of thetwo day (48 hour) observation, demonstrating the highly acute effects ofcyanide overexposure.

For completion of the studies, a rat model (Sprague-Dawley) ofsubcutaneous (S.C.) exposure to KCN was employed. For all experiments,KCN was dissolved in normal saline such that a volume between 0.2 and1.0 ml would be injected. As with the parathion experiments,post-injection data was compared to control data obtained prior toinjection of KCN for each animal. In addition, a group of animals wasgiven 1.0 ml saline S.C. as an added control group. These studies wereperformed with a modified fundoscope for imaging the internal regions ofthe rat eye under non-mydriatic conditions. Due to the requirement ofhaving the animals remain very still during recording, long termanesthesia with a ketamine/xylazine mixture (80 mg/kg ketaminehydrochloride; 12 mg/kg xylazine hydrochloride) was induced prior torecording.

The experimental procedure was as follows: animals were weighed,anesthetized with ketamine/xylazine, and then placed on an adjustableplatform in position for proper fundoscopic imaging. Approximately 10-15minutes after anesthetic induction, a needle (23-gauge) attached to a 1ml syringe containing KCN was inserted under the skin and between therear shoulder blades of the animal. Baseline fundoscopic imaging wasthen continually recorded. Five minutes after the beginning ofrecording, the plunger on the lml syringe was depressed, subcutaneouslyinjecting the animal with KCN. Images were continually recorded for 60minutes or until death (whichever came first). Example images from oneof the animals are shown in FIG. 4.

Subsequent to obtaining streaming images, individual images from thestreaming video were isolated at specific time periods. Vesselcoloration was then quantitatively analyzed. Arteries and veins wereidentified and subsequently isolated based on characteristicpresentation (caliber, location, etc.). When possible, A-V pairs(adjacent artery and vein) were evaluated to minimize anyillumination-induced measurement errors. In order to complete theanalysis, a number of different digital signal processing (DSP) methodswere examined and tested for optimizing the discrimination capabilitiesof the system while accounting for the expected variability betweenindividuals (in this case animals). Initial analysis was conducted usingvery simplistic processing algorithms. This involved simply convertingthe images to a gray scale, determining the average coloration of thevessel of interest (0-255), and then using these values to determine themeans and standard errors for subsequent analysis. Via these preliminaryanalytical methods, the lowest level of subcutaneous KCN that was foundto yield statistically significant differences was 1.0 mg/kg.

In an attempt to increase the discriminatory capabilities of thevasculature analysis protocol, additional signal processing techniqueswere implemented and evaluated. Working with gray scale images greatlyenhanced the efficiency of analysis and interpretation of effects. Inaddition, statistical comparison of the linear output values of the grayscale was more convenient for the current goal of establishing venousblood coloration as a characteristic for cyanide exposure. Though use ofthe full color range to analyze images provides greater sensitivity(16.8 million colors vs. 256 gray scale colors), such analysis alsorequires a much more complex three-dimensional (RGB; red-green-blue)comparison.

Incorporating color images with automated algorithmic analysis couldprovide a much more powerful discriminatory ability, but is not requiredfor the present purposes. For that reason, a relatively non-cumbersomeroute for the manual analysis was used. The process involved (1)converting the color images to gray-scale (0-255), (2) flattening theillumination characteristics, and (3) using histogram equalization toimprove contrast in the color range which the vessels reside. All ofthese functions were performed using the commercially available product,Corel® Draw™ 11 (Corel Corporation, Ottawa, Ontario, Canada). Onceconversion was completed, the original image, processed image, andanalytical values were archived. Employing these basic processingtechniques allowed for relatively rapid analysis times whilesignificantly enhancing the sensitivity of the measurements for cyanideexposure diagnosis. This improved sensitivity was evidenced bydecreasing the KCN dose which yielded statistically significantobservable differences from 1.0 mg/kg (as above) to 30 μg/kg (seebelow), a 30-fold enhancement of diagnostic capabilities.

After exposure to KCN, the onset of altered blood coloration,generalized exposure signs/symptoms, and death (after large lethaldoses) is quite rapid, even with the subcutaneous route of application.As seen in FIG. 5, in response to 1 mg/kg KCN (s.c.), arterial vesselcoloration did not change significantly (though there was a slight trendtoward brighter coloration) whereas venous coloration brightenedsignificantly within 60 seconds of cyanide injection (p<0.05).

These alterations in venous coloration/oxygenation were often quicklyfollowed by generalized symptoms such as rapid respiration withincreased depth of breath. Maximal changes in venous coloration weretypically noted within five minutes of KCN administration. FIG. 6 plotsthe time course of venous coloration change in response to variousconcentrations of I.P. KCN.

For construction of the concentration-response relationship between KCNand venous coloration, the 5 minute post-administration time point waschosen for analysis. As noted above, all changes in venous colorationreached a maximum level by this time point and only animals which wereadministered supramaximal lethal doses of 30 mg/kg or greater expiredwithin this time frame. Curve fitting analysis of theconcentration-response curve shown in FIG. 7 for venous colorationchange 5 minutes after subcutaneous KCN injection estimated an EC₅₀ formaximal detected venous color change of 24.6±3.1 μg/kg. However,statistically significant changes were not seen in every animal examineduntil the 30 μg/kg test group (p<0.05).

In addition to the approximate 200-fold diagnostic window (LD₅₀=6.7mg/kg; significant venous changes at 30 μg/kg) provided by venousalterations, it also became apparent that there may be some diagnosticutility in the analysis of arterial coloration. We found a slight butsignificant increase in the color value of the arteries at greater KCNdosage levels. This change became significant at the 300 μg/kg level(p<0.05). Since the arterial coloration does not appear to be assensitive a diagnostic indicator for KCN exposure it may be useful as asecondary indicator for exposure level, much as the pupillary lightreflex appears to be useful for organophosphate exposure. Thus, anindication of significant coloration changes (to brighter, moreoxygenated levels) in both arterial and venous systems indicates agreater level of exposure than venous changes alone. Furthermore, itshould be noted that only animals which displayed changes in bothsystems (arteries and veins) later died as a result of their exposure.Thus, in mass casualty situations this may provide a valuablediscriminating tool to identify individuals who have been exposed tosublethal levels of cyanide versus those who have been exposed topotentially lethal levels of the compound. With more advanced signalprocessing methods, this discriminatory capability may be furtherincreased.

Example 4 Exposure to Carbon Monoxide

As was done for each of the agents previously discussed, an initialanalysis of the lethal effects of CO was performed. The nature of CO,namely that it is a gas, places such experiments more at the discretionof the researcher. Whereas with chemicals/toxins a standard singleinjection LD₅₀ study may be employed, with inhalational agents there isthe added variable of application time. A 48-hour administration of COis not feasible (would require continual animal monitoring) and there isno standard protocol for such tests. Thus, reported values for lethallevels of various gases/gas mixtures vary with respect to bothconcentration of gas and length of application. Often these reportedvalues will be for lethal effects ensuing after 4 or 8 hours of gasadministration. However, since the protocol used for the studiesdescribed here employ a 60 minute observation window, the 1 hour timepoint was used for the lethality determination endpoint. The 60 minuteexposure LD₅₀ determined in the present studies was 3123±476 ppm(parts-per-million).

In addition to changes in lethality testing analysis, the route ofexposure (inhalation) mandates changes to the dosing protocol.Typically, as evidenced in dosing patterns plotted in figures forprevious agents, single injection agents are dosed based on the normallogarithmic concentration-response characteristics. Thus a dosingpattern of 1, 3, 10, 30, etc. is normally employed to give a symmetricallog-dose comparison. However, the compounding effect of continual gasexposure leads to a toxic load which makes such dosing patternsimpractical for proper quantitative analysis, as they will not yieldconcentration-response curves which sensitively and accurately reflectthe actions of the gas (i.e., the dose ranges are too broad/diffuse).For this reason, variable increments in carbon monoxide percentage wereused in the present experiments, concentrating primarily around theregion of initially observable significant effects.

For completion of the studies, a rat model (Sprague-Dawley) ofinhalational exposure to CO was used. As with the previous experiments,post-exposure data was compared to control data which was obtained priorto CO administration for each animal. In addition, a group of animalswas connected to the inhalational exposure apparatus and administeredcompressed normal air (79% nitrogen, 21% oxygen) as an added controlgroup. Imaging studies were performed with a modified fundoscope forimaging the internal regions of the rat eye under non-mydriaticconditions. Due to the requirement of having the animals remain verystill during recording, long term anesthesia with a ketamine/xylazinemixture (80 mg/kg ketamine hydrochloride; 12 mg/kg xylazinehydrochloride) was induced prior to recording.

Experimental procedures were as follows: animals were weighed,anesthetized with ketamine/xylazine, and then placed on an adjustableplatform in position for proper fundoscopic imaging. The animals werethen connected to the inhalation apparatus and allowed to breathe normalair. Approximately 10 minutes after initial anesthetic induction,baseline fundoscopic images were continually recorded. Approximately 15minutes after anesthetic induction, a valve was adjusted to switch theanimal's breathing mixture from 79% N₂/21% O₂ to a predetermined mixtureincluding CO (or, in control cases, a separate normal air mixture). Dueto the logistics of preparing a large range of CO doses, pure CO wasmixed with appropriate levels of compressed normal air, resulting inproportional decreases in both N₂ and O₂ partial pressures/compositionpercentages. Images were continually recorded for 60 minutes or untildeath, whichever came first.

Data procurement and analysis was done in an identical manner as forKCN-exposed animals. Briefly, streaming images were obtained, individualimages were isolated from the streaming video, images were processedwith basic DSP methods and then quantitatively analyzed for vesselcoloration. Arteries and veins were identified and subsequently isolatedbased on characteristic presentation (caliber, location, etc.) and, whenpossible, A-V pairs (adjacent artery and vein) were evaluated tominimize any illumination-induced measurement errors. Digital signalprocessing methods used for these images were the same as those chosenfor final implementation with KCN-exposed animals: (1) the color imageswere converted to gray-scale (0-255), (2) illumination characteristicswere flattened, and (3) histogram equalization was employed to improvecontrast in the color range which the vessels reside.

As the time course of vessel coloration change is examined in responseto an approximate 60 minute LD₅₀ dose (3000 ppm), two characteristicsbecome evident. Firstly, even at this high level of CO administration,the alteration in blood coloration is relatively gradual and linear.This fact is likely due to the route of administration and mechanism ofaction of CO. The change in coloration is proportional to the toxic loadof CO, which increases in an approximate linear fashion with time.Secondly, statistically significant changes can be observed in botharterial (becomes significant at 15 minutes; p<0.05) and venous (becomessignificant at 30 minutes; p<0.05) blood vessels. Interestingly, just asevidenced for the 3000 ppm dose in FIG. 9, significant changes in venouscoloration trailed (occurred later in time) significant changes inarterial coloration at each concentration of CO which caused significantchanges in both vessel types within the 60 minute application period(i.e., 60 minute doses of 500 ppm and greater). This indicates that agreater CO load is required to produce significant changes in venousblood coloration. This differential sensitivity to CO dosing may allowvessel coloration analysis to additionally indicate total CO load inaddition to simply exposure vs. non-exposure.

Careful examination of the following CO dose-response curve providesadditional information with respect to the functional diagnosis of COinhalation. First, near maximal changes (92.3%) in arterial colorationwere detectable in animals which did not expire within the 60 minutetreatment period. Thus, either: (1) prolonged exposure to the maximaleffects of CO are required prior to animal expiration, or (2) there is athreshold level in arterial oxygenation changes beyond which the presentanalytical methods cannot distinguish further changes in coloration.Secondly, statistically significant changes (p<0.05) were detected inanimals which received 250 ppm CO for 60 minutes, 12-fold below LD₅₀levels (3123 ppm). Unfortunately, this indication of a “diagnosticwindow” is not as indicative as it may be for other agents, for reasondescribed above concerning exposure times. Though there is a twelve folddifference for the 60 minute exposure time, this ratio may likely varyas the exposure time is varied. However, based on the foregoing it canbe noted with confidence that diagnosis of CO exposure is clearlyobtainable at levels of exposure far less than lethal levels.

Example 5 Exposure to Botulinum Toxin

The first task was to determine the LD₅₀ for botulinum toxin. An initialsurvey of the literature as well as the internet (Centers for DiseaseControl, MSDS sheets, etc.) did not reveal the existence of priordeterminations of the botulinum toxin LD₅₀ in rats. Therefore,preliminary dosing levels were based on the LD₅₀ reported for mice, 1ng/kg. In the present study, employing intraperitoneal (I.P.) injectionof botulinum toxin Type A, an extrapolated LD₅₀ of 2.28±0.97 ng/kg wasobtained. If greater than 48 hours post-injection had been used as theendpoint for these experiments, this value would likely have been muchlower. Many of the animals which received doses 10- to 30-fold lowerthan our 48 hour LD₅₀ (2.28 ng/kg) displayed advanced motor symptomologyby the 48 hour post-injection time point, including decreased grossmotor movements, jerky/uncoordinated movements, and labored breathing.Such signs were found to be consistent indicators of impending animalexpiration. Even at the highest dose of botulinum toxin administered (30μg/kg), over 68,000 times the 48 hour LD₅₀, animal expiration did notoccur until at least 14 hours post-injection.

Regardless of dose, however, the onset and progression of symptomsfollowed a very similar and consistent pattern: initial increase inchewing behavior, decreased exploratory behavior, qualitative overallmuscle weakness, jaw clenching and labored breathing, followed withinhours by animal expiration. This was also evident with the initialocular characteristic investigated, pupil motility. Thus, both the timeof onset and the magnitude of pupil motility decrease were dependentupon the concentration of botulinum toxin administered. In order todetermine the most appropriate time(s) to do the primary dosecomparisons, the time course of changes in the pupillary light reflexand how such changes related temporally to the onset of othersymptoms/death were investigated.

Sprague-Dawley rats were weighed, briefly anesthetized with halothane,and then administered the appropriate I.P. dose of Type A botulinumtoxin (Metabiologics, Madison, Wis.). The toxin was diluted in saline toa concentration which allowed administration of between 0.2 and 1.0 mlof solution. Control animals were given 1 ml of saline I.P. in the samemanner. Animals were subsequently tested for pupil size and pupillarylight reflex in 1 hour increments post-injection in the same manner asin the parathion experiments discussed earlier. Briefly, animal pupilswere monitored and recorded under very low light intensity conditions(1.7 cd/m²). For pupillary light reflex measurements, the animals weresubjected to “steps” in light intensity for response analysis. For eachtime point indicated, this involved 1 minute of high intensity lighting(80 cd/m²) followed by return to baseline low level lighting intensity.

Experiments were designed and conducted in a “staggered” manner suchthat each animal was not actually tested for each of the 48 hourspost-injection. Instead, by staggering the dosing schedule it wasinsured that there were sufficient numbers of animals at each dose toprovide 4 measurements for each time point (hour) without the need formonitoring and testing each animal for a continual 48 hours. These timecourse data are presented in FIG. 11.

The most consistently observed generalized motor symptom was loss ofexploratory behavior. This was descriptively quantified when an animal,upon being placed into a new cage for observation, did not move from thecenter location where it was placed within 1 minute. Turning/rotatingmovements were considered exploratory even if the animal remained in thecenter of the cage. A “positive” score for this test (i.e., no animalmovement) was used to denote generalized motor symptomology. This testwas performed hourly, just prior to ocular examinations.

Construction of a concentration-response curve for inhibition of thepupillary light reflex subsequent to botulinum toxin injection was notfeasible for these experiments. Due to the highly varying rates of onset(i.e., onset occurs in low dose animals well after death occurs in highdose animals) a standard measurement time for which effects were nearmaximal in all animals was not obtainable. However, some indication asto the diagnostic capabilities of light reflex impairment may still beascertained. In all animals which eventually expired, diminishedpupillary light reflexes were detected. The time at which these deficitswere detectable varied. The lowest botulinum toxin dose which resultedin statistically significant decreases in the pupillary light reflex was10 pg/kg (p<0.05), 200-fold below the LD₅₀. Thus, it can be determinedthat the pupillary light reflex is a sensitive indicator (with regard todose and time) of botulinum intoxication. It is, however, apparent thatthe ability to detect poisoning is highly dependent upon both theconcentration of botulinum toxin administered as well as the length oftime since administration.

In an attempt to enhance the diagnostic capabilities of pupil motilityfor the detection of botulinum toxin exposure a separate study wasdeveloped. The aim of these studies was to investigate whetherphysiological challenge of the pupillary light reflex with strobelighting could more finely discern differences between exposed andnon-exposed animals. In normal animals there is a maximal frequency atwhich the pupillary light reflex can function. This frequency is limitedby the rate at which impulses denoting a light flash can travel from theretina, through the pupillary light reflex loop, and back to the musclescontrolling pupil size for effect. In humans this maximal frequency isapproximately 5 Hz (loop takes approximately 200 msec). The maximalfrequency in normal Sprague-Dawley rats is about 3.48±0.57 Hz. Thus,significant decreases in the maximal response frequency afterover-stimulation with high intensity light flashes can be indicatedegradations along the reflex loop and may identify possible botulinumtoxin exposure.

After pupil size and pupillary light reflex were tested during each timepoint as described above, the following protocol was used to examine thehigh intensity over-stimulation effect. Five minutes after conclusion ofpupil size/reflex testing, the animal was subjected to a high intensity(90 cd/m²) light flashing at a frequency of 1 Hz using a Grass photicstimulator tuned with universal counter-timer (Global SpecialtiesCorporation). This frequency was incrementally stepped (0.1 Hz/step)every five seconds while continually recording pupillary fluctuations at30 Hz via the video imaging system. Stepping continued until a frequencyof 4 Hz was obtained (40 steps over 200 seconds). Preliminary studiesindicated that 4 Hz was sufficiently above the maximal frequency atwhich normal rat pupils could respond to these light flashes. Dataanalysis then involved frame-by-frame analysis of the recorded images.The frequency at which the pupil ceased oscillating in size (in responseto the intermittent flashes of light) and instead remained at aconstant, fixed size as determined by the initial flash at a givenfrequency was determined to be the animals “cutoff” frequency. As notedabove, this value for normal rats was 3.48±0.57 Hz.

The results of this study indicate that the experimental determinationof a strobe cutoff frequency may be used for diagnostic determination ofbotulinum toxin exposure. However, much like earlier studies of thepupillary light reflex, the presentation time of these deficits werevariable in relation to administered dose. As illustrated in thefollowing Graph 11, the strobe cutoff frequency was temporally moresensitive than the normal pupillary light reflex test in diagnosingbotulinum exposure (significantly more sensitive with concentration of100 ng/kg and lower, p<0.05). Though this test of pupillary motilitydoes increase the time window (prior to general symptomology and death)for diagnosis of botulinum toxin exposure, there remains the importantvariable of analysis time beyond that which exists for the other agents.Implementation of the described characteristics would require repeateddiagnostic checks to insure that the observation time is not too soonafter potential exposure. However, as with current clinical diagnosis,similar problems are likely to exist for any diagnostic evaluation ofbotulinum toxin exposure.

Example 6 Diagnosis of Exposure/Non-Exposure to Unknown

Due to administration and/or temporal characteristics unique to eachagent, special care was taken to ensure the blinded validity of thesestudies. Since different routes of administration were necessary(parathion and botulinum toxin intraperitoneally, potassium cyanidesubcutaneously, and carbon monoxide via inhalation) and different timepoints post-administration were used for data collection, 6 replicatesof 3 concentrations of each agent as well as matched controls were used.This design preserved the ability to mask the data collector as to thepredicted outcome (i.e., if only one dose of CO was used without matchedcontrols, observer would know treatment based upon inhalationaladministration). Upon collection, data was given to a secondexperimenter who analyzed, without prior designation as to treatment,the data by transferring it to a Microsoft® Excel worksheet and runningdiagnostic macros. Use of these macros allowed automated analysis of thedata, removing analytical judgment, bias, and/or error from thediagnostic determination. Data with animal diagnoses were then returnedto the experimenter who knew the treatment (and level) for statisticalanalysis.

A flow chart illustrating an exemplary diagnostic pathway is presentedin FIG. 1. The “cutoff” levels for the normal/control range weredetermined identically for each step. These ranges represent the controlanimal mean values ±2 standard deviations as determined during theexperiments previously discussed. The blinded study included diagnosticevaluation of 100 Sprague-Dawley rats. Animals were administered testagent or control agent as detailed in Table 1, below.

TABLE 1 Animal composition for the blinded study. Potassium CarbonBotulinum Parathion Cyanide Monoxide Toxin (intraperitoneal)(subcutaneous) (inhalation) (intraperitoneal) 1 μg/kg 3 μg/kg 250 ppm 30pg/kg (n = 6*) (n = 6) (n = 6) (n = 6) 10 μg/kg 30 μg/kg 750 ppm 300pg/kg (n = 6) (n = 6) (n = 6) (n = 6) 1 mg/kg 3 mg/kg 3500 ppm 3 ng/kg(n = 6) (n = 6) (n = 6) (n = 6) 1 ml Corn Oil 1 ml Saline Normal Air 1ml Saline (n = 7)^(†) (n = 7) (n = 7) (n = 7) *Number of animals at eachconcentration identified in parentheses. ^(†)Bottom row lists the 4control groups which were included (administered by same route asagent).

As noted in Table 1, three concentrations of each agent were included.These doses were chosen based on previous EC₅₀ and LD₅₀ determinationsas well as minimal discernable effects. An approximate LD₅₀ dose wasused for the highest dose, an approximate EC₅₀ dose (for the ocularcharacteristic) was used as the medium/mid dose, and a “minimal effect”dose was given as the lowest dose.

The diagnostic paradigm included two levels of evaluation: (1) diagnosisof exposure to a specific agent vs. non-exposed, and (2) if diagnosed asexposed, determination of the approximate level of exposure (low-, mid-,or high-level, corresponding to the three dosage levels). The first“level” of evaluation served as the primary test for ocularcharacteristic diagnostic validity to determine whether agents can beselectively indicated via ocular examination. The second “level” ofevaluation provided an indicator of the sensitivity of the ocularcharacteristics for discerning gradations of exposure, as describedabove. Such capabilities may be valuable in mass casualty scenarioswhere both diagnosis of exposure type and level of need are importantdeterminants. The results of the blinded, automated diagnosis of theseanimals are presented in Table 2.

TABLE 2 Results of automated diagnosis of animals from 4 treatmentgroups (parathion, potassium cyanide, carbon monoxide, botulinum toxin)and matched controls including 3 subgroups for each agent. Correct AgentCorrect Agent and Level Exposure & Level Diagnosis/Total (%)Diagnosis/Total (%)* Control 28/28 (100%)^(#)   N/A 1 ml Corn Oil (i.p.)7/7 (100%) N/A 1 ml Saline (s.c.) 7/7 (100%) N/A Normal Air Inhaled 7/7(100%) N/A 1 ml Saline (i.p.) 7/7 (100%) N/A Parathion (i.p.) 16/18(89%)   16/18 (89%)   1 μg/kg 4/6 (67%)  4/6 (67%)  10 μg/kg 6/6 (100%)6/6 (100%) 1 mg/kg 6/6 (100%) 6/6 (100%) Potassium Cyanide (s.c.) 15/18(83%)   14/18 (78%)   3 μg/kg 3/6 (50%)  3/6 (50%)  30 μg/kg 6/6 (100%)5/6 (83%)  3 mg/kg 6/6 (100%) 6/6 (100%) Carbon Monoxide (inhaled) 16/18(89%)   15/18 (83%)   250 ppm 4/6 (67%)  3/6 (50%)  750 ppm 6/6 (100%)6/6 (100%) 3500 ppm 6/6 (100%) 6/6 (100%) Botulinum Toxin (i.p.) 12/18(67%)   11/18 (61%)   30 pg/kg 0/6 (0%)  0/6 (0%)  300 pg/kg 6/6 (100%)6/6 (100%) 3 ng/kg 6/6 (100%) 5/6 (83%)  TOTALS 87/100 (87%)   56/72(78%)   *Indicates proportion of animals correctly diagnosed withrespect to both the agent and the level of agent. ^(#)Proportion andpercentage for all levels of a given agent exposure

All non-exposed, mid-level-exposed, and high-level-exposed animals werecorrectly diagnosed with respect to agent (76 of 76, 100%). Of these,only 2 animals were misidentified with respect to exposure level (1mid-level KCN-exposed diagnosed as low-level KCN exposure and 1high-level botulinum toxin-exposure animal diagnosed as mid-levelbotulinum exposure). All fully missed diagnoses were at low levels ofagent, with the many of these (6 of 13 total, 46%) being low-levelbotulinum toxin-exposed animals. The portion (11 of 24, 46%) of thelow-level-exposed animals which were correctly diagnosed with respect toexposure was much greater than was anticipated. The concentrations ofthe various agents used for these low-level exposures were below levelswhich caused significant changes during the experiments previouslydiscussed. Thus, the fact that nearly half of these animals werecorrectly diagnosed indicates a greater level of diagnostic sensitivitythan was expected. In addition, analysis of the statistical data fromthe blinded study indicates that the paradigm used to make thediagnostic decisions may be improved for greater discernment of thelow-level agents. In particular, an increased incorporation of low-lightpupil size and the pupillary light reflex data into the botulinum toxindiagnosis may enhance our ability to correctly identify animals exposedto low levels of this agent.

CONCLUSION

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. It will be understood by those skilledin the art that various changes in form and details may be made thereinwithout departing from the spirit and scope of the invention. Thus, thebreadth and scope of the invention should not be limited by any of theabove-described exemplary embodiments.

1. A method of diagnosing cyanide exposure of a subject, comprising thestep of: (a) determining whether the subject's ocular blood vesselcoloration varies significantly from a normal ocular blood vesselcoloration by: (1) quantifying the subject's ocular blood vesselcoloration; and (2) comparing the quantity from said step (1) to aquantified normal vessel coloration.
 2. The method of claim 1, whereinthe normal ocular blood vessel coloration of said step (a) is determinedfor each subject.
 3. The method of claim 1, wherein the normal ocularblood vessel coloration of said step (a) is determined as an average fora population to which the subject belongs.
 4. The method of claim 1,wherein the ocular blood vessels of said step (a) are venous vessels. 5.The method of claim 1, wherein the ocular blood vessels of said step (a)are arterial vessels.
 6. A method of diagnosing carbon monoxide exposureof a subject, comprising the step of: (a) determining whether thesubject's ocular blood vessel coloration varies significantly from anormal ocular blood vessel coloration by: (1) quantifying the subject'socular blood vessel coloration; and (2) comparing the quantity from saidstep (1) to a quantified normal vessel coloration.
 7. The method ofclaim 6, wherein the normal ocular blood vessel coloration of said step(a) is determined for each subject.
 8. The method of claim 6, whereinthe normal ocular blood vessel coloration of said step (a) is determinedas an average for a population to which the subject belongs.
 9. Themethod of claim 6, wherein the ocular blood vessels of said step (a) arevenous vessels.
 10. The method of claim 6, wherein the ocular bloodvessels of said step (a) are arterial vessels.
 11. A method ofdiagnosing botulinum toxin exposure of a subject, comprising the stepof: (a) determining whether the subject's strobe cutoff frequency variessignificantly from a normal strobe cutoff frequency by: (1) quantifyingthe subject's strobe cutoff frequency; and (2) comparing the quantityfrom said step (a1) to a quantified normal strobe cutoff frequency. 12.The method of claim 11, wherein the normal strobe cutoff frequency ofsaid step (a) is determined for each subject.
 13. The method of claim11, wherein the normal strobe cutoff frequency of said step (a) isdetermined as an average for a population to which the subject belongs.